Evidence suggests that lipid membrane interactions with rare, broadly neutralizing antibodies (NAbs), 2F5 and 4E10, play a critical role in HIV-1 neutralization. The objective of this proposal is to understand the polyreactivity of NAbs, specifically how lipid membrane properties, such as composition, lipid domain organization, and lipid diffusivity contribute to 2F5/4E10 membrane interactions and antigen localization at the membrane interface, with the ultimate vision of guiding immunogen designs. Recent immunization studies have shown that induction of antibodies that avidly bind the gp41-MPER antigen is not sufficient for neutralization. Rather, it is required that antigen designs induce polyreactive antibodies that can recognize MPER antigens as well as the viral lipid membrane. However, the mechanistic details of how membrane properties influence NAb-lipid and NAb-antigen interactions remain largely unknown. To understand how membrane properties contribute to 2F5/4E10 membrane interactions, we have engineered supported lipid bilayers (SLBs) whose compositions mimic the HIV-1 envelope (Env). We hypothesize that (1) NAbs, 2F5/4E10, and HIV-1 antigen, MPER656, exclusively associate with the most fluid membrane domains. The composition, diffusivity, and surface energies of these domains dictate NAb and antigen interactions with lipid membranes. (2) CDR H3 loops on 2F5 and 4E10 embed selectively into mobile domains on HIV-1 Env mimetic membranes. This ability of CDR H3 loops to anchor into the lipid bilayer gives rise to a preferential antibody binding orientation that reslts in extraction of membrane-submerged antigen residues. To test these hypotheses, we have two specific aims: (SA1) i) Identify the localization of NAbs and antigen on model membrane surfaces. ii) Determine the membrane characteristics that help drive NAb and antigen interactions with lipid domains; i.e., membrane diffusivity, domain composition, and domain surface energies. (SA2) i) Identify binding depth and orientation of 2F5/4E10 on SLBs that mimic mobile domains of HIV-1 lipid Env and ii) identify conformations of NAb-antigen binding at the membrane interface. Our research is significant in that it will reveal molecular details of th role of lipid membranes underlying 2F5/4E10 antigen binding. It is suggested that induction of polyreactive antibodies that can bind to both lipids and MPER antigen is required for neutralization. A recent attempt to use MPER peptide liposomes with only phospholipid components of the host cell membrane failed to induce polyreactive MPER antibodies, although MPER epitope specific antibodies were induced. Importantly, the liposomal design in the above study did not include key anionic lipid components to which 2F5 and 4E10 bind, and also did not include some of the viral lipid components that are abundant on HIV-1 virions. Thus, it is the objective of this proposal to provide information for new immunogen designs that incorporate, in addition to the gp41 MPER epitope, key viral lipid components to which 2F5 and 4E10 bind. The antigen paired with an optimal lipid environment is more likely to induce the required polyreactive antibodies.